Salts enhance both protein stability and amyloid formation of an immunoglobulin light chain

Amyloid fibrils are associated with sulfated glycosaminoglycans in the extracellular matrix. The presence of sulfated glycosaminoglycans is known to promote amyloid formation in vitro and in vivo, with the sulfate groups playing a role in this process. In order to understand the role that sulfate plays in amyloid formation, we have studied the effect of salts from the Hofmeister series on the protein structure, stability and amyloid formation of an amyloidogenic light chain protein, AL-12. We have been able to show for the first time a direct correlation between protein stability and amyloid formation enhancement by salts from the Hofmeister series, where SO₄²⁻ conferred the most protein stability and enhancement of amyloid formation. Our study emphasizes the importance of the effect of ions in the protein bound water properties and downplays the role of specific interactions between the protein and ions.     

Mycorrhizal synthesis between <Emphasis Type="Italic">Boletus edulis</Emphasis> species complex and rockroses (<Emphasis Type="Italic">Cistus</Emphasis> sp.)

Ectomycorrhizas of Boletus aereus, Boletus edulis, and Boletus reticulatus were synthesized with Cistus sp. under laboratory conditions using synthesis tubes filled with a mixture of sterilized peat-vermiculite and nutrient solution. The fungal strains isolated from sporocarps were identified by molecular techniques. The inoculated seedlings were grown for 4–5 months. The ectomycorrhizas formed were described based on standard morphological and anatomical characters. The three ectomycorrhizas described were very similar, with white monopodial-pinnate morphology, a three-layered plectenchymatous mantle on plan view and boletoid rhizomorphs.     

Partial persistence of exercise-induced myocardial angiogenesis following 4-week detraining in the rat

Enhanced angiogenesis, or capillary growth, has a prominent role among the various beneficial effects of exercise training on the myocardium. The aim of the present study is to assess if training-induced increases in capillarity and vascularization persist after 4 weeks of detraining. Adult male rats were trained to run on a treadmill for 10 weeks at ∼60% VO_2max, which did not induce cardiac hypertrophy, but increased (P < 0.05) the soleus/body weight ratio, left ventricle capillarity and von Willebrand-positive cell density (n = 6). In another group of animals (n = 6) subjected to training followed by 4-week detraining, the soleus/body weight ratio returned to normal, with only partial reversal of left ventricle capillarity and von Willebrand-positive cell density. Markers of angiogenesis (VEGF, KDR/VEGF-R2 and HIF-1α mRNA, studied by real-time RT-PCR) were upregulated at the end of training, and returned to baseline value after detraining. Electron microscopy highlighted some morphological features in trained hearts (endothelial cell sprouting and bridges and pericyte detachment), suggestive of endothelial cell proliferation and capillary growth that were absent in untrained and detrained hearts. We conclude that the training-induced increase in cardiac capillarity and vascularization are retained for some time upon cessation of the training program even in the absence of angiogenic stimuli.     

Thin semitransparent gels containing phenylboronic acid: porosity, optical response and permeability for sugars

Radical copolymerization of acrylamide (Am) (90 mol%) with N-acryloyl-m-aminophenylboronic acid (NAAPBA) (10 mol%) carried out on the surface of glass slides in aqueous solution and in the absence of chemical cross-linkers, resulted in the formation of thin semitransparent gels. The phenylboronic acid (PBA) ligand density was ca. 160 micromol/ml gel. The gels exhibited a macroporous structure and displayed optical response to sucrose, lactose, glucose and fructose in 50 mM sodium phosphate buffer, in the pH range from 6.5 to 7.5. The response was fairly reversible and linearly depended on glucose concentration in the wide concentration range from 1 to 60 mM at pH 7.3. The character of response was explained by the balance of two competing equilibrium processes: binding of glucose to phenylboronate anions and binary hydrophobic interactions of neutral PBA groups. The apparent diffusion coefficient of glucose in the gels was ca. 2.5 x 10(-7) cm(2)/s. A freshly prepared gel can be used daily for at least 1 month without changes in sensitivity. Autoclaving (121 degrees C, 1.2 bar, 10 min) allows for the gels sterilization, which is important for their use as glucose sensors in fermentation processes.     

Dimeric transferrin inhibits phagocytosis of residual bodies by testicular rat Sertoli cells

Transferrin is well known as an iron transport glycoprotein. Dimeric or tetrameric transferrin forms have recently been reported to modulate phagocytosis by human leukocytes. It is mainly synthesized by the liver, and also by other sources, such as Sertoli cells of the testis. Sertoli cells show a strong phagocytic activity toward apoptotic germ cells and residual bodies. Here, we provide evidence that purified human dimeric transferrin from commercial sources decreased residual body phagocytosis, unlike monomeric transferrin. The presence of iron appeared essential for dimeric transferrin inhibitory activity. Importantly, dimeric transferrin could be visualized by immunoblotting in Sertoli cell lysates as well as in culture media, indicating that dimeric transferrin could be physiologically secreted by Sertoli cells. By siRNA-mediated knockdown, we show that endogenous transferrin significantly inhibited residual body ingestion by Sertoli cells. These results are the first to identify dimeric transferrin in Sertoli cells and to demonstrate its implication as a physiological modulator of residual body phagocytosis by Sertoli cells.     

Production of recombinant proteins in clonal root cultures using episomal expression vectors

We have developed a fully contained system for expressing recombinant proteins that is based on clonal root cultures and episomal expression vectors. Clonal root lines expressing green fluorescent protein (GFP) or human growth hormone were generated from Nicotiana benthamiana leaves infected with the tobacco mosaic virus-based vector 30B after exposure to Agrobacterium rhizogenes. These lines accumulated GFP at over 50 mg per kg fresh tissue, a level that is comparable with other plant production systems in early stage development. Accumulation of both hGH and GFP in the clonal root lines was sustained over a 3-year period, and in the absence of antibiotic selection. This technology shows promise for commercial production of vaccine antigens and therapeutic proteins in contained facilities.     

FAK-independent alphavbeta3 integrin-EGFR complexes rescue from anoikis matrix-defective fibroblasts

Extracellular matrix (ECM) binding to integrin receptors regulates cell cycle progression and survival. In adherent cells, ECM disassembly induces anoikis, the apoptotic pathway switched on by loss of adhesion. ECM-deficient Ehlers-Danlos syndrome (EDS) fibroblasts, to adhere to rare fibronectin (FN) fibrils, and to proliferate, only organize, as FN receptor, the alphavbeta3 integrin. We report that in EDS cells the alphavbeta3 integrin is bound to talin and vinculin, but not to tensin, and that actin cytoskeleton is disorganized. Furthermore, in EDS cells Bcl-2 is down-regulated and caspases are active. We provide evidence that the antibody-mediated alphavbeta3 integrin or the FN inhibition induces anoikis in EDS cells. The alphavbeta3 integrin transduces survival signals to pp60src-mediated tyrosine phosphorylated paxillin, instead than to FAK, and interacts with EGF receptor (EGFR). This complex, when activated by EGF and FN, signals for the rescue of EDS cells from anoikis. Therefore, EDS cells, through the alphavbeta3 integrin-EGFR complexes, engage a paxillin- but not FAK-mediated pathway of cell survival.     

PKCepsilon mediates glucose-regulated insulin production in pancreatic beta-cells

Endocrine cells produce large amounts of one or more peptides. The post-translational control of selective production of a single protein is often unknown. We used 3 unrelated approaches to diminish PKCepsilon in rat islets to evaluate its role in preferential glucose-mediated insulin production. Transfection with siRNA (siR-PKCepsilon) or expression of inactive PKCepsilon (PKCepsilon-KD) resulted in a significant reduction in insulin response to glucose (16.7 mmol/l). Glucose stimulation resulted in concentration of PKCepsilon in the perinuclear region, an area known to be rich in ER-Golgi systems, associated with insulin-containing structures. ss'COP1 (RACK2) is the anchoring protein for PKCepsilon. Glucose-stimulated proinsulin production was diminished by 50% in islets expressing PKCepsilon-KD, and 60% in islets expressing RACK2 binding protein (epsilonV1-2); total protein biosynthesis was not affected. In islets expressing epsilonV1-2, a chase period following glucose stimulus resulted in a reduced proinsulin conversion to mature insulin. We propose that PKCepsilon plays a specific role in mediating the glucose-signal into insulin production: binding to ss'COP1 localizes the activated enzyme to the RER where it modulates the shuttling of proinsulin to the TGN. Subsequently the enzyme may be involved in anterograde trafficking of the prohormone or in its processing within the TGN.     

Green algae in tundra soils affected by coal mine pollutions

Green algal communities were investigated in clean and pollution-impacted tundra soils around the large coal mine industrial complex of Vorkuta in the E. European Russian tundra. Samples were collected in three zones of open-cast coal mining with different degrees of pollution-impacted soil transformation. A total of 42 species of algae were found in all zones. The species richness decreased from 27 species in undisturbed zones to 19 species in polluted zones. Under open-cast coal mining impacts the community structure simplified, and the dominant algae complexes changed. Algae that are typical for clean soils disappeared from the communities. The total abundance of green algae (counted together with Xanthophyta) ranged between 100–120 × 10³ (cells/g dry soils) in undisturbed zones and 0.5–50 × 10³ in polluted zones. Soil algae appear to be better indicators of coal mine technogenic pollution than flowering plants and mosses. Presented at the International Symposium Biology and Taxonomy of Green Algae V, Smolenice, June 26–29, 2007, Slovakia.     

Immunotherapy of neuroblastoma by an Interleukin-21-secreting cell vaccine involves survivin as antigen

AIM: IL-21 is the most recently identified member of the IL-2 cytokine family. Here we studied the therapeutic efficacy of IL-21-gene-modified cells (Neuro2a/IL-21) in a syngeneic metastatic neuroblastoma (NB) model. MATERIALS AND METHODS: Neuro2a/IL-21 cells were tested as subcutaneous (sc) vaccine both in prophylactic and therapeutic settings. Depletion studies, cytotoxicity assay and immunohistochemical analyses were carried out to evaluate the mechanisms involved in tumor rejection. RESULTS: When injected sc in syngeneic A/J mice viable Neuro2a/IL-21 cells were rejected and induced resistance to a subsequent iv challenge with Neuro2a parental cells (Neuro2a/pc), suggesting the involvement of an immune response. More importantly, in mice bearing Neuro2a/pc micrometastases, a single sc injection of Neuro2a/IL-21 cells significantly increased the mean tumor-free survival of treated animals (43 vs. 22 days) and cured 14% of them. The administration of two or three doses of Neuro2a/IL-21 cell vaccine further increased the mean survival time to 54 and 75 days, and the cure rate to 27 and 33%, respectively, whereas the use of unmodified Neuro2a or mock-transfected cells had no effect. In vivo cell subset depletion and a Winn-assay indicated the involvement of CD8 + CTLs. Immunohistochemical analysis indicated a reduction of CD31+ and VEGFR2+ microvessels in late metastases from therapeutically vaccinated mice. A role of survivin as antigen was suggested by in vitro assays using survivin-synthetic CTL-epitopes. CONCLUSIONS: Our present data indicate that IL-21-secreting NB cells are effective as therapeutic vaccine in mice bearing metastatic NB, through a specific CTL response involving survivin as antigen, and suggest a potential interest for IL-21 in NB immuno-gene therapy.